1. Field of the Invention
The invention is generally directed to degradative enzyme systems. In particular, the present invention is directed to chitin depolymerases and associated proteins and enzymes found in Microbulbifer degradans. 
2. Background of the Invention
Chitin, a homopolymer of repeating units of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc), is the second most abundant polymer in the biome. It is found in various forms throughout the marine environment and is a component of crustacean and insect exoskeletons, yeast and fungal cell walls, and diatoms. Chitin is usually at least 90% acetylated and is often in a complex with proteins and other carbohydrates. The microcrystalline structure of chitin varies between antiparallel sheets (alpha chitin), parallel sheets (beta chitin), and a mixture of both (gamma chitin). Alpha chitin is found in the calyces of hydrozoa, mollusks, plankton, and as a component of the cuticles of arthropods. Beta chitin, a less stable and more degradable form of chitin, is found in mollusks, squid pen, diatoms, and insect exoskeletons and cocoons, and is the major component of fungal cell walls.
Microbulbifer degradans strain 2-40 is a marine γ-proteobacterium that was isolated from decaying Sparina alterniflora, a salt marsh cord grass in the Chesapeake Bay watershed. Consistent with its isolation from decaying plant matter, M. degradans strain 2-40 is able to degrade many complex polysaccharides, including cellulose, pectin, xylan, and chitin, which are common components of the cell walls of higher plants. M. degradans strain 2-40 is also able to depolymerize algal cell wall components, such as agar, agarose, and laminarin, as well as protein, starch, pullulan, and alginic acid. In addition to degrading this plethora of polymers, M. degradans strain 2-40 can utilize each of the polysaccharides as the sole carbon source. Therefore, M. degradans strain 2-40 is not only an excellent model of microbial degradation of insoluble complex polysaccharides (ICPs) but can also be used as a paradigm for complete metabolism of these ICPs. ICPs are polymerized saccharides that are used for form and structure in animals and plants. They are insoluble in water and therefore are difficult to break down.
Chitin is a difficult substrate for microbial degradation because it is usually crystalline and complexed with protein, salts, and other carbohydrates. Chitin is resistant to chemical degradation and is difficult to digest enzymatically because of the multiple steps required to expose and cleave the polymer. Because chitin resists chemical and physical breakdown, microorganisms must play a major role in its degradation. Many microorganisms have developed efficient strategies for the depolymerization, transport, and metabolism of chitin and its derivatives. These systems involve multiple enzyme activities, usually encoded on separate polypeptides. For example, Pseudoalteromonas strain S91, Serratia marcescens, and Streptomyces coelicolor secrete several chitin-depolymerizing enzymes in the presence of chitin. Surprisingly, almost no free chitin is found in marine sediments, demonstrating the efficiency of these microbial systems. Therefore, chitin represents an abundant source of carbon and nitrogen to microorganisms in the marine environment.
The glycoside hydrolase family 18 (GH18) domain is the most common catalytic domain of microbial chitin depolymerases. Despite sharing a consensus sequence and a conserved catalytic glutamic acid residue, GH18 domains differ in their activity toward polymeric chitin and chito-oligosaccharides (i.e., endo- versus exo-activity). Chitodextrinases, which depolymerize chitooligosaccharides but not chitin, also contain GH18 domains. Chitinolytic enzymes with GH18 domains have been isolated from organisms as diverse as psychrophilic eubacteria and hyperthermophilic archaeons, demonstrating the wide range of conditions to which these domains have adapted. Because conserved residues are found in GH18 domains with divergent optima and substrate specificities, sequence analysis is insufficient to determine the enzymatic specificities of newly discovered chitinases.
Endo- and exo-chitinases that function cooperatively to depolymerize chitin are known. Endochitinases randomly cleave glycosidic linkages, generating free ends and long chitooligosaccharides. These are then acted upon by exochitinases that release chitobiose from the non-reducing ends of each. While exo- and endo-chitinases are not able to depolymerize chitin alone, the presence of both activities significantly increases the efficiency of chitinolytic systems.
Therefore, there exists a need to identify enzyme systems that use chitin as a substrate, express the genes encoding the proteins using suitable vectors, identify and isolate the amino acid products (enzymes and non-enzymatic products), and use these products as well as organisms containing these genes to degrade plant and animal waste.